This is an application for renewal of a grant to study the structure, function and mechanism of the viral RNA-dependent RNA polymerase (RdRP). Positive-strand RNA viruses cause a variety of diseases in humans. Strategies to control disease outbreaks caused by this category of viruses are needed. The central hypothesis driving the proposed studies is that the viral RdRP is a tractable target for development of broad-spectrum antiviral agents. The previous funding period was devoted to the elucidation of the kinetic, thermodynamic and structural basis for nucleotide incorporation catalyzed by a prototypical RdRP, 3Dpol from poliovirus that would permit a quantitative, mechanistic comparison of the viral RdRP to cellular polymerases. All of the original aims were completed. During the next funding period, we will pursue the following specific aims: (1) Mechanistic studies of 3Dpol-catalyzed nucleotide incorporation. We hypothesize that a conformational change preceding phosphoryl transfer is a key determinant of 3Dpol fidelity. We propose to develop innovative methodologies to evaluate this step directly. We have identified conditions for the 3Dpol reaction in which phosphoryl transfer is the sole rate-limiting step. This advance permits us to interrogate the chemical mechanism directly. We will test the hypotheses that one of the two metal ions required for catalysis functions to orient the 3' nucleophile of the primer for catalysis and that phosphoryl transfer employs general acid-base catalysis. A conformational change exists after phosphoryl transfer. We hypothesize that this step represents another fidelity checkpoint. We will refine existing approaches and develop new approaches to study this step directly. (2) Structural basis for fidelity of 3Dpol-catalyzed nucleotide incorporation. We will employ a novel genetic selection strategy to isolate poliovirus mutants with an antimutator phenotype in order to uncover structural determinants of fidelity. This approach has already provided a link between a site remote from the catalytic center and the conformational change preceding phosphoryl transfer, an unexpected yet exciting finding. We will continue to test predictions of our structural model for the 3Dpol ternary complex in order to define all of the residues participating in the hydrogen-bonding network in the ribose-binding pocket that is required for incorporation fidelity. We will test the hypothesis that a loop of unknown function and unique to RdRPs and reverse transcriptases located in the palm subdomain of 3Dpol is required for incorporation fidelity. (3) Determinants of 3Dpol-primer/template complex stability. A crystal structure for a ternary complex of 3Dpol does not exist. If we can assemble 3Dpol-primer/template complexes that do not dissociate on the timescale required for crystallization, then we will be in a better position to fill this gap. We will pursue two approaches that may permit us to achieve this goal, and, at the same time, provide insight into the mechanisms and surfaces employed by 3Dpol to maximize stability of the 3Dpol-primer/template complex.